POS0235 ANTI-INFLAMMATORY EFFECTS OF TIP-1 AND MIP-2 TREATMENT THROUGH TLRS BLOCKADE IN AOSD ANIMAL MODEL
Authors: M. H. Ahn, Y. S. Choi, S. W. Lee, S. Choi, H. A. Kim
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Adult-onset Still's disease (AOSD) is a rare systemic inflammatory disease manifesting with a typical skin rash, hepatosplenomegaly, persistent high fever, and leukocytosis. There is no suitable animal model to research for AOSD. The signaling of Toll-like receptors (TLR) as pattern-recognizing receptors is necessary for pathogen recognition by the innate immunity. Recent studies have shown the TLR-mediated inflammation could be an important role in pathogenesis of AOSD. TLR inhibitor peptide 1 (TIP-1) and MAL/MyD88 inhibitory peptide 2 (MIP-2) have been evaluated with blocking peptide activity to bind TIR domain, which is recognition of the danger- or pathogen-associated molecular patterns (DAMP or PAMP). <h3>Objectives</h3> This study tried to evaluated that these inhibitory peptides could suppress their inflammation and improve clinical manifestations in animal models of AOSD. <h3>Methods</h3> <i>Mycobacterium</i> mixed Freund's complete adjuvant (CFA, 1.5 mg/ml) was subcutaneously injected into IFNγ knock-out (IFNγ KO) mice to inducing AOSD. TIP-1 or MIP-2 (10 nmol/g) was intraperitoneally administered every day for 4 weeks. Fever and body weight were checked every week. The size of lymph node (LN) and spleen was measured when the animals were sacrificed. The level of IL-1β and IL-18 in serum were measured by ELISA. Downstream of TLRs signal pathway, MyD88 and NFκB, were confirmed by WB. <h3>Results</h3> <i>Mycobacterium</i> mixed CFA injected IFNγ KO mice showed a significantly higher fever than normal mice (<i>p</i>= 0.0009). These fever were significantly reduced after TLR inhibitory peptide treatment (TIP-1; <i>p</i>=0.008, MIP-2; <i>p</i>=0.029). In addition, hypertrophy of the spleen and LN was found in IFNγ KO mice, which were statistically significantly improved through TLR inhibitor injection (TIP-1 and MIP-2, <i>p</i>&lt;0.05). The level of IL-1β and IL-18 in serum were significantly increased in the CFA injected IFNγ KO mice and increased inflammatory cytokines was decreased by TLR inhibition (TIP-1 and MIP-2, <i>p</i>&lt;0.05). MyD88 and phosphorylated NFκB were increased in LN of CFA injected IFNγ KO mice and the proteins was down-regulated with treatment of TIP-1 or MIP-2. The IL-1β expression in LN and spleen was increased in CFA injected IFNγ KO mice and it decreased in TIP1 or MIP-2 treated mice. <h3>Conclusion</h3> Abnormal fever, lymph node and spleen tissue enlargement, and increase in serum inflammatory cytokines (IL-1β, IL-18), etc., have been confirmed that the symptoms accompanying adult-type Still's disease appear in animals. In addition, it was confirmed that these immune responses were reduced in the TLR inhibitor treatment group. Collectively, these in vivo findings mean that <i>Mycobacterium</i> mixed CFA injected IFNγ KO mice was showed pathological features of AOSD and TIP-1and MIP-2 has broad specificity for TLRs and should be effective plausible candidate for the treatment of AOSD. <h3>References</h3> [1]Jung JY KJ, Suh CH, Kim HA: Roles of Interactions Between Toll-Like Receptors and Their Endogenous Ligands in the Pathogenesis of Systemic Juvenile Idiopathic Arthritis and Adult-Onset Still's Disease. Front Immunol 2020, 11:583513. [2]Shah M KG, Achek A, Cho EY, Baek WY, Choi YS, Lee WH, Kim DJ, Lee SH, Kim W, Kim SS, Cheong JY, Suh CH, Choi S: The αC helix of TIRAP holds therapeutic potential in TLR-mediated autoimmune diseases. Biomaterials 2020, 245:119974. [3]Achek A KH, Patra MC, Shah M, Hong R, Lee WH, Baek WY, Choi YS, Kim GY, Pham TLH, Suh CH, Kim W, Hahm DY, Choi S: A peptide derived from the core β-sheet region of TIRAP decoys TLR4 and reduces inflammatory and autoimmune symptoms in murine models. EBioMedicine 2020, 52:102645. [4]Baek WY CY, Lee SW, On IO, Jeon KW, Choi S, Suh CH: Toll-like Receptor Signaling Inhibitory Peptide Improves Inflammation in Animal Model and Human Systemic Lupus Erythematosus. Int J Mol Sci 2021, 22:12764. [5]Vandenhaut J AA, Filtjens J, Malengier-Devlies B, Imbrechts M, Van den Berghe N, Ahmadzadeh K, Mitera T, Boon L, Leclercq G, Wouters C, Matthys P.: Regulatory Role for NK Cells in a Mouse Model of Systemic Juvenile Idiopathic Arthrits. J Immunol 2019, 203:3339-3348. <h3>Acknowledgements</h3> This work was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology [grant number 2022R1I1A2065168 and 2019R1A2C1005988], Korea. <h3>Disclosure of Interests</h3> None Declared.